EP1722624B1 - Procede pour produire des piments transgeniques par callogenese - Google Patents

Procede pour produire des piments transgeniques par callogenese Download PDF

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EP1722624B1
EP1722624B1 EP04748401.9A EP04748401A EP1722624B1 EP 1722624 B1 EP1722624 B1 EP 1722624B1 EP 04748401 A EP04748401 A EP 04748401A EP 1722624 B1 EP1722624 B1 EP 1722624B1
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callus
pepper
explants
medium
zeatin
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EP1722624A4 (fr
EP1722624A1 (fr
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Chee Hark Harn
Yun Hee Lee
Ju Yeon Kim
Hyo Soon Kim
Min Jung
Soon Ho Choi
Seung Gyun Yang
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NONGWOOBIO
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/005Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205Agrobacterium mediated transformation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance

Definitions

  • the present invention relates to a method for producing transgenic pepper plants, and more particularly to a method for mass-producing transgenic pepper plants using callus induction, the method being characterized by the steps of: pre-culturing pepper explants; co-culturing the pre-cultured pepper explants with Agrobacterium into which a target gene have been introduced; selecting callus; and forming shoots.
  • Pepper is one of the main crops in the world and widely used as food or seasonings. Also, pepper is used in industrial dyes, particularly it is used as a main raw material of cosmetic lipsticks. The total annual sales amount of pepper products in Korea is about two trillion won in Korean currency and forms the largest seed market in Korea. Pepper is the most suitable crop to be able to create high added value in relation to the use of biotechnology.
  • the first gateway to biotechnically use pepper is to perform transformation.
  • pepper is known as a crop which is very difficult to be transformed due to the following two reasons.
  • the infection rate of Agrobacterium varies depending on crops. It is inferred that crops, which are difficult to be transformed, are the case where the pilus of Agrobacterium is impermeable to the tissue cell of explants or not sufficiently inoculated to the tissue cell.
  • transgenic pepper can be easily obtained, thereby completing the present invention.
  • a main object of the present invention is to provide a method for mass-producing transgenic pepper plants, the method being characterized by pre-culturing explants of pepper in callus induction medium, followed by co-culturing with Agrobacterium.
  • the present invention provides a method for mass-producing transgenic pepper plants, the method comprising the steps of: (a) pre-culturing explants of pepper in a callus induction medium; (b) co-culturing the pre-cultured explants with Agrobacterium into which a target gene have been introduced; (c) culturing the co-cultured explants in a selection medium so as to form callus and selecting the formed callus; and (d) cutting the callus and culturing the cut callus in a shoot induction medium so as to form shoots.
  • explants refers to tissue segments excised from plants, which includes cotyledons or hypocotyls.
  • the method of the present invention comprises the following steps: (a) the pre-culture of pepper explants; (b) transformation (co-culture with Agrobacterium into which a target gene have been introduced), (c) callus selection; and (d) shoot formation.
  • the inventive method may additionally comprise the steps of root formation and soil adaptation.
  • the inventive method is characterized by that pepper explants are pre-cultured in callus induction medium and transformed so as to artificially form callus, and the regeneration of plants is induced from the formed callus.
  • explants of pepper are first pre-cultured in callus induction medium. At this time, the explants are preferably wounded in order to promote callus induction. Furthermore, as the pepper explants, cotyledons or hypocotyls can be used in the present invention. Cotyledons obtained by sterilizing and washing pepper seeds and germinating the washed seeds in MS medium are more preferably used.
  • the callus induction medium which is used in the pre-culture step of the inventive method refers to a plant culture medium (e.g., MS medium and 1/2 MS medium) containing a plant hormone that induces callus.
  • the plant hormone which can be used for callus induction is one selected from the group consisting of zeatin, 2,4-dichlorophenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA), naphthalene acetic acid (NAA) and benzylaminopurine (BA).
  • Each of the plant hormones is added at the following concentration: 0.01-5 mg/l of 2,4-D, 0.01-5 mg/l of IAA, 0.01-5 mg/l of zeatin, 0.01-5 mg/l of NAA, and 0.01-5 mg/l of BA. Most preferably, 1 mg/l of 2,4-D or 1 mg/l of IAA may be used. A mixture of the above-mentioned hormones may also be used. Preferably, a mixture of 0.01-2 mg/l of zeatin with one selected from the group consisting of 0.1-5 mg/l of 2,4-D, 0.1-5 mg/l of IAA and 0.01-2 mg/l of NAA may be used.
  • the pre-culture step is preferably performed at 22-28°C for 20-60 hours. During the pre-culture step, callus is induced at the wounded parts of the explants by the influence of the hormone.
  • the pre-cultured explants are co-cultured with Agrobacterium.
  • a target gene to be introduced into pepper plants needs to be introduced into Agrobacterium.
  • the transformation of Agrobacterium may be performed by any method known in the art. For example, the freeze-thaw method ( Glevin et al., Plant molecular biology. Kluwer Academic Publishers A3/7, 1988 ) may be used.
  • Agrobacterium into which the target gene have been introduced is inoculated to the pre-cultured pepper explants for 10-20 minutes and then co-cultured.
  • the same medium as the callus induction medium used in the step (a) is preferably used as co-culture medium.
  • the co-culture step is preferably performed at 22-28°C for 40-80 hours.
  • the pepper explants which have been co-cultured with Agrobacterium are cultured in selection medium to select transgenic callus.
  • the selection medium preferably contains not only an antibiotic for the selection of transgenic callus but also zeatin and IAA for the induction of callus development. As a substitute for IAA, NAA may also be used.
  • the antibiotic used in the callus selection is preferably an antibiotic corresponding to an antibiotic-resistant gene present in a recombinant vector introduced into Agrobacterium.
  • zeatin is preferably used at a concentration of 0.1-5 mg/l
  • each of IAA and NAA is preferably used at a concentration of 0.01-1 mg/l.
  • a mixture of 2 mg/l of zeatin and 0.3 mg/l of IAA may be used. 4-5 weeks after the explants co-cultured with Agrobacterium are transferred to the selection medium, callus begins to grow. Then, the callus is preferably cultured for additional 5-8 weeks.
  • the transformed callus obtained in the step (c) is cut into small pieces.
  • the cut callus pieces are transferred to shoot induction medium to induce shoot formation.
  • the shoot induction medium preferably contains zeatin alone or a mixture of zeatin and one of IAA and NAA.
  • Zeatin is preferably used at a concentration of 0.5-10 mg/l, and each of IAA and NAA is preferably used at a concentration of 0.01-0.2 mg/l. More preferably, a mixture of 2 mg/l of zeatin and 0.01 mg/l of IAA may be used.
  • One month after the callus is transferred to the shoot induction medium, shoots are formed. Then, the shoots are preferably cultured in the same medium for an additional period of about two months, so as to elongate the shoots.
  • the elongated shoots are transferred and cultured in root induction medium.
  • the root induction medium preferably contains no plant hormone.
  • a MS basal medium containing a small amount of antibiotics may be used as the root induction medium.
  • roots are formed. When the roots grow about 4-10 cm, the medium is clearly removed and the roots are planted in pots.
  • the inventive method for transforming pepper plants has the following advantages.
  • the inventive method is not limited to certain lines of pepper plants (line non-specific). Thus, the inventive method can stably transform various lines of pepper plants.
  • the inventive method can mass-produce transgenic plants within a shorter time period than that of the prior method, since the callus induction hormone is added in the step of pre-culturing the explants so as to shorten the time for callus induction.
  • various useful genes such as genes involved in plant defense mechanisms or genes involved in the biosynthesis of useful metabolites, can be introduced into pepper plants by the inventive transformation method, so that a variety of functional transgenic pepper plants can be mass-produced at high efficiency.
  • Example of the genes involved in plant defense mechanisms are preferably genes encoding a protein selected from the group consisting of ribosome inactivating protein (RIP), jasmonic acid carboxyl methyltransferase, trehalose synthase, plant defensin 1.2, thionine synthase, glucanase, chitinase, phenylalanine ammonia lyase, chalcone synthase, glutathione- S -transferase, anthranilate synthase, storage protein, calmodulin, tryptophan synthase, proteinase inhibitor II, nitric oxide synthase, cystemine, fatty acid peroxidase, allene oxide synthase, pepper-PMMV interaction 1 transcription factor (PPI1), WRKY domain transcription factor, pathogen-responsive protein, and virus coat protein, but are not limited thereto.
  • RIP ribosome inactivating protein
  • genes involved in the biosynthesis of useful metabolites are preferably selected from the group consisting of genes involved in the biosynthesis of tannin, sinapine, saponin, allicin, spinosin, cinnamic acid, flavonoid, terpenoid, catechin, vitamin, penicilline, indole, insulin, prostaglandin, taxol, alisol, ricin, cartenoid and capsisin, but are not limited thereto.
  • Capsicum annuum L. examples include chilli pepper (Capsicum annuum L. var. acuminatum), sweet or bell pepper (Capsicum annuum L. var. grossum), cone pepper (Capsicum annuum L. var. conoides), cherry pepper (Capsicum annuum L. var. cerasiforme), red cluster pepper ( Capsicum annuum L. var. fasciculatum), long pepper (Capsicum annuum L. var. longum), and so on.
  • explants of pepper were pre-cultured in a medium containing either a mixture of zeatin and NAA or a mixture of zeatin and IAA, and then transformed with Agrobacterium into which a TMV-CP gene or a PPI1 gene have been introduced. Thereafter, unlike a general case where shoots are formed directly from wounded parts of transformed explants during a regeneration process of pepper (direct shoot formation), it was observed that there was a case where callus had been induced in wounded parts, from which shoots have then been formed (indirect shoot formation; callus-mediated shoot formation) (see FIG. 1 and FIG. 2 ).
  • the present invention provides transgenic pepper plants transformed with a gene encoding a PPI1 protein, as well as TMV-resistant pepper plants transformed with a gene encoding TMV-CP.
  • the pepper explants were pre-cultured in a medium containing one or more of various plant hormones and then co-cultured with Agrobacterium into which a GFP gene have been introduced.
  • Agrobacterium into which a GFP gene have been introduced.
  • 2,4-D, IAA or a mixture of zeatin and 2,4-D were used as the plant hormone, respectively.
  • the transformed explants were cultured in selection medium to select transgenic callus (see FIG. 3 and FIG. 4 ).
  • the callus was cut into small pieces and then transferred to shoot induction medium to form shoots (see FIG. 5 ). Following this, roots were induced from the shoots and then regenerated into complete plants via an adaptation step (see FIG. 6 ).
  • the callus induction rates in the case where the pepper explants have been pre-cultured in the medium containing a mixture of 2,4-D and zeatin, 2,4-D or IAA was examined. The results showed that the callus was generally induced at a rate of about 13%, although there is a slight difference in callus induction rate between pepper lines (see Table 6 ). This callus induction rate is about 10 times higher than that of the case where explants were pre-cultured in a medium containing zeatin and NAA (or IAA). Furthermore, the transformation rate of shoots formed from callus was about 1%, based on the total number of explants used in transformation (see FIG 7 ).
  • This value is about 5 times higher than the callus-mediated shoot transformation rate (0.19%) of the case where explants were pre-cultured in a medium containing zeatin and NAA (or IAA). Moreover, in the case where the explants were pre-cultured in the medium containing the mixture of 2,4-D and zeatin, 2,4-D or IAA, it was found that all of 8 line peppers used in a test were stably transformed (see Table 6 and FIG. 7 to FIG. 9 ). The inventive method as described above allowed the transformation time to be shortened as compared to the prior methods by pre-culturing the pepper explants in the callus induction medium.
  • Example 1 Production of pepper plants transformed with TMV-CP gene or PPI1 gene
  • a DNA fragment encoding a TMV-CP gene (GenBank accession No: L35074; Park et al., 1997) or a PPI1 gene (GenBank accession No: AF430372; Lee et al., 2002) was inserted into a pCAMBIA 2300 vector (CAMBIA, Australia).
  • the resulting recombinant vector was introduced into an Agrobacterium LBA4404 strain or EHA105 strain.
  • the transformed Agrobacterium strain was cultured in a YEP medium containing 50mg/l kanamycin, 50mg/l rifampicin and 100 ⁇ M acetosyringone. The culture broth was centrifuged and then diluted in MS basal medium to an OD 600 of 0.3-0.5.
  • the culture suspension was mixed with a MS basal medium containing 100 ⁇ M acetosyringone, and the mixture was inoculated to the explants pre-cultured in Example 1-1 above for 10-20 minutes. Thereafter, the explants were co-cultured with the Agrobacterium strain in a medium having the same composition as that of the pre-culture medium used in Example 1-1 above, under dark conditions for 38-96 hours. The explants co-cultured with the Agrobacterium strain were washed three times with a 1/2 MS liquid medium containing 500 ⁇ 800 mg/l cefotaxime or lilacilline.
  • the explants co-cultured with the Agrobacterium strain in Example 1-2 above were cultured in a MS basal medium (selection medium) containing 2 mg/l zeatin, 0.05 mg/l NAA (or 0.1 mg/l IAA), 80-100 mg/l kanamycin and 300 mg/l cefotaxime (or lilacilline) for 6-8 weeks. 4-5 weeks after the culture, it could be observed that tissue similar to callus was formed around the wounded parts of several explants.
  • the explants were transferred to a MS basal medium (elongation medium) containing 2 mg/l zeatin, 0.01 mg/l NAA (or 0.01 mg/l IAA), 60-100 mg/l kanamycin and 300mg/l cefotaxime and cultured for 7-10 weeks.
  • MS basal medium elongation medium
  • the elongated shoots were transferred to a MS basal medium (root induction medium) containing 20-30 mg/l kanamycin and 200 mg/l cefotaxime and cultured for 6-8 weeks.
  • the regenerated plants were transferred into a zippy pot and cultured under a 16 hr light condition at 25°C for 2 weeks.
  • One pattern is a general pattern where shoots (or multiple shoots) are formed directly from the wounded parts of the explants (direct shoot formation), and the other pattern is one where callus tissue is formed around the wounded parts of the explants, and then, shoots are formed from the callus tissue (indirect shoot formation; callus-mediated shoot formation).
  • the regeneration process by the direct shoot formation was observed in the most cases.
  • the regeneration process consisted of 5 steps including shoot formation, multi-shoot formation, multi-shoot elongation, single-shoot elongation, and root formation.
  • the regeneration process by the indirect shoot formation mediated by callus was very rarely observed, and this is believed that it is because of callus not being easy to be naturally induced from the wounded parts of cotyledons.
  • the regeneration process by the indirect shoot formation consisted of five steps including callus formation, callus development, shoot formation, single-shoot elongation and root formation.
  • Table 1 Frequency of direct shoot formation Genes Number of explants Number of shoots Number of shoots with root P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101 TMV-CP 30,512 26,039 24,107 21,983 2,106 1,017 1,697 628 392 156 311 49 PPI1 14,413 14,080 7,488 13,078 1,024 587 720 310 186 103 176 34 Subtotal 44,925 40,119 31,595 35,061 3,130 1,604 2,417 938 578 259 487 83 Total 151,700 8,089 1,407
  • a total of 37,500 explants obtained from four lines of pepper plants were transformed in the same manner as in Example 1 above, after which the frequency of callus-mediated shoot formation was examined.
  • the frequency of callus formation from the explants was a very low level of 1.2% (459/37,500) (see Table 2 ).
  • the frequency of shoot development from the callus was 11.6% (53/459), and the frequency of root formation from the shoots was 52.8% (28/53).
  • Table 2 Frequencies of callus development and shoot formation from callus Genes Number of explants Number of callus formed Number of shoots formed from the callus Number of shoots with roots P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101 P915 P409 P410 P101 TMV-CP 5,188 6,917 5,491 7,210 81 30 25 94 14 5 1 7 12 3 0 2 PPI1 2,903 3,056 3,798 2,937 107 46 17 59 15 4 2 5 7 2 1 1 1 Sub-total 8,091 9,973 9,289 10,147 188 76 42 153 29 9 3 12 19 5 1 3 Total 37,500 459 53 28
  • genomic DNA of pepper plants was isolated according to the method described in Lee et al., Plant Mol. Biol., 46:661, 2001 .
  • PCR was performed using primers represented by SEQ ID NO: 1 and SEQ ID NO: 2.
  • PCR was performed by repeating 35 cycles, each consisting of 1 minute at 94°C, 1 minute at 55°C and 1 minute at 72°C.
  • T0 plants were self-crossed to obtain T1 plants.
  • the leaves of 408 individuals of the T1 plants were inoculated with TMV twice at intervals of two-weeks.
  • ELISA was performed using a TMV-CP antibody ( Shin, R. et al., Mol. Planet Microbe. Interact., 15:983, 2002 ).
  • the results showed that 28 individuals of 408 individuals of the T1 plants were resistant to TMV infection (see Table 5 ).
  • Mosaic spots were observed in the leaves of susceptible plants, but not observed in the resistant plants.
  • the explants were pre-cultured in a medium containing one or more of various plant hormones.
  • the cotyledons were wounded with a knife, after which they were transferred to a callus induction medium containing one or more of various plant hormones (MS basal medium containing 1 mg/l 2,4-D, 1 mg/l IAA or a mixture of 1 mg/12,4-D and 0.2 mg/l zeatin) and pre-cultured.
  • MS basal medium containing 1 mg/l 2,4-D, 1 mg/l IAA or a mixture of 1 mg/12,4-D and 0.2 mg/l zeatin
  • the pre-culture was performed in a temperature-controlled incubator (22-28°C) under light conditions for 20-60 hours.
  • a GFP gene (GenBank accession No: AY508125; Haseloff J, et al., PNAS, 94:2122, 1997 ) was introduced into a pCAMBIA 2300 vector.
  • the resulting recombinant vector was introduced into an Agrobacterium LBA4404 strain.
  • the transformed Agnobacterium strain was cultured in a YEP medium containing 50mg/l kanamycin, 50mg/l rifampicin and 100 ⁇ M acetosyringone. The culture broth was centrifuged, and then diluted in MS basal medium to an OD 600 of 0.3-0.6.
  • the culture suspension was mixed with a MS liquid medium containing 100 ⁇ M acetosyringone, and the mixture was inoculated to the cotyledons pre-cultured in Example 5-1 above for 20 minutes. Thereafter, the cotyledons were co-cultured with the Agrobacterium strain in a medium having the same composition as that of the pre-culture medium used in Example 5-1, under dark conditions for 48-70 hours. The cotyledons co-cultured with the Agrobacterium strain were washed three times with a 1/2 MS liquid medium containing 500-800 mg/l cefotaxime.
  • the cotyledons co-cultured with the Agrobacterium strain in Example 5-2 above were transferred to a MS basal medium (selection medium) containing 2 mg/l zeatin, 0.3 mg/l IAA, 80 mg/l kanamycin, 100 mg/l cefotaxime and 300 mg/l lilacilline, and cultured in an incubator under a 16-hr light/8-hr dark cycle for 4-5 weeks. During this culture process, callus began to grow.
  • FIG. 3 is a photograph showing the callus grown for two weeks after transfer to the selection medium. Then, the callus was additionally cultured for 5-8 weeks.
  • the growth pattern of the transgenic callus according to the culture time was observed under UV microscope and the results are shown in FIG. 4 . As shown in FIG. 4 , it could be confirmed that the transgenic callus expressing GFP shows continuous expression of GFP during its growth, thereby indicating that it is genetically stable.
  • the grown callus was cut into small pieces, and then transferred to a MS basal medium (shoot induction medium) containing 2 mg/l zeatin, 0.01 mg/l IAA, 30-60 mg/l kanamycin and 300 mg/l cefotaxime and cultured for about one month. Thereafter, the formed shoots were additionally cultured for about two months so as to elongate the formed shoots.
  • the process of shoot formation from the transformed callus is shown in FIG. 5 .
  • the shoots were cultured in a MS basal medium containing 20-30 mg/l kanamycin and 200 mg/l cefotaxime. After about 4-5 weeks, roots were formed from the shoots. When the roots grew about 10 cm, the medium was removed and the remaining roots were planted in a zippy pot. They were cultured in an incubator for a few days, and then cultured in a greenhouse. The appearance of transgenic pepper plants obtained one month after the roots were planted in the zippy pot is shown in FIG. 6 .
  • Genomic DNAs were extracted from callus expressing GFP and callus expressing no GFP under UV, respectively, among the transgenic plants obtained in Example 5 above.
  • each of the extracted DNA templates was subjected to PCR using primers represented by SEQ ID NO: 5 and SEQ ID NO: 6.
  • PCR was performed by repeating 35 cycles, each consisting of 1 minute at 60°C, 1 minute at 94°C and 1 minute at 72°C.
  • the PCR assay results showed that a band with a size of about 720bp corresponding to the GFP gene was detected only in the callus expressing GFP.
  • a genomic DNA was extracted from the leaves of several lines of seedlings regenerated from the callus expressing GFP, and then subjected to PCR in the same manner as described just above. As shown in FIG. 8 , the PCR assay results showed that the GFP gene was stably introduced into all of P410, P915, P318 and P319 lines. This indicates that the transformation method according to the present invention is not line-specific.
  • Genomic DNAs were extracted from 14 individuals of the transgenic pepper plants (T0) obtained in Example 5 above, and then subjected to Southern blot analysis, thus determining the copy number of a GFP gene inserted into the transgenic plants.
  • Example 5 The callus induction in 8 lines of pepper plants was attempted in the same manner as in Example 5. The results showed that callus was induced in all the lines at a general rate of about 13%, although there was a difference in callus induction rate between the pepper plant lines (see Table 6 ). Particularly, the case of treatment with 2,4-D or IAA alone showed a higher callus induction rate than that of the case of treatment with a mixture of 2,4-D and zeatin. Furthermore, the callus induction rate of Example 5 was about 10 times higher than that of Example 1 (1.2%; see Table 2 ).
  • Table 6 Transformation rate according to callus induction rate and GFP expression Pepper lines Number of explants Number of callus induced (ratio, %) Number of callus expressing GFP (ratio, %) P915 1343 202(15.0) 68(5.1) P318 853 27(3.2) 9(1.1) P319 596 186(31.2) 82(13.8) P409 933 77(8.3) 27(2.9) P410 402 76(18.9) 19(4.7) P784 56 5(8.9) 2(3.6) P2377 312 43(13.8) 22(7.1) PMAL 415 29(7.0) 4(1.0) Total 4910 645(13.1) 233(4.7)
  • the frequency of shoot formation from the callus induced from explants was examined for about 5 months after co-culture with Agrobacterium, and the result showed a shoot formation frequency of about 37.7% (see Table 7 ) .
  • PCR assay on the shoots formed from callus was performed to examine the introduction rate of a GFP gene.
  • the PCR assay results showed that the ratio of shoots having a GFP gene insertion among the shoots formed from callus was 45% ( Table 7 ).
  • the transformation rate of the shoots with the GFP gene was determined to be about 1% (27/2792) based on the total number of explants used in transformation. This value is about 5 times higher than that of Example 1 (0.19%) (see Table 4 ).
  • Table 7 Shoot formation rate and transformation rate from callus Pepper lines Number of shoots formed from the callus Number of shoots positive in the PCR assay P915 12/68 2 P318 8/9 4 P319 40/82 21 Total 60/159 (37.7%) 27/60 (45.0%)
  • the pepper explants were pre-cultured in callus induction medium and then transformed so as to artificially form callus, and the regeneration of plants was induced from the callus.
  • the inventive method shows very high transformation efficiency and can transform pepper plants in a line non-specific manner. Furthermore, the inventive method allows mass-producing various lines of transgenic pepper plants at high efficiency, since the time taken for the preparation of the transgenic pepper plants is shortened as compared to the prior method.

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Claims (18)

  1. Procédé de production de plants de piment transgéniques, le procédé comprenant les étapes suivantes :
    (a) pré-culture d'explants de piment dans un milieu d'induction de cals ;
    (b) co-culture des explants pré-cultivés avec Agrobacterium chez lequel un gène cible a été introduit ;
    (c) culture des explants co-cultivés dans un milieu de sélection de façon à former un cal et sélection du cal formé ; et
    (d) excision du cal et culture du cal excisé dans un milieu d'induction de pousses de façon à former des pousses.
  2. Procédé selon la revendication 1, dans lequel les explants de l'étape (a) sont blessés afin de promouvoir l'induction de cals.
  3. Procédé selon la revendication 1, dans lequel les explants de l'étape (a) sont des cotylédons ou des hypocotyles.
  4. Procédé selon la revendication 1, dans lequel le milieu d'induction de cals de l'étape (a) comprend un ou plusieurs agents d'induction choisis dans le groupe constitué par la zéatine, l'acide 2,4-dichlorophénoxy-acétique (2,4-D), l'acide indole-3-acétique (IAA), l'acide naphtalène acétique (NAA) et la benzylaminopurine (BA).
  5. Procédé selon la revendication 4, dans lequel le milieu d'induction de cals de l'étape (a) comprend un ou plusieurs agents d'induction choisis dans le groupe constitué par 0,01-5 mg/l de 2,4-0, 0,01-5 mg/l d'IAA, 0,01-5 mg/l de zéatine, 0,01-5 mg/l de NAA et 0,01-5 mg/l de BA.
  6. Procédé selon la revendication 4, dans lequel le milieu comprend 1 mg/l de 2,4-D ou 1 mg/l d'IAA.
  7. Procédé selon la revendication 4, dans lequel le milieu comprend un mélange de 0,01-2 mg/l de zéatine avec un agent d'induction choisi dans le groupe constitué par 0,1-5 mg/l de 2,4-D, 0,1-5 mg/l d'IAA et 0,01-2 mg/l de NAA.
  8. Procédé selon la revendication 1, dans lequel la co-culture dans l'étape (b) est réalisée dans le même milieu que le milieu d'induction de cals utilisé dans l'étape (a).
  9. Procédé selon la revendication 1, dans lequel le milieu de sélection dans l'étape (c) comprend un mélange de 0,1∼5 mg/l de zéatine et de 0,01∼1 mg/l d'IAA ou un mélange de 0,1~5 mg/l de zéatine et de 0,01~1 mg/l de NAA.
  10. Procédé selon la revendication 1, dans lequel le milieu d'induction de pousses dans l'étape (d) comprend un agent d'induction choisi dans le groupe constitué par :
    (i) 0,5~10 mg/l de zéatine ;
    (ii) un mélange de 0,5~10 mg/l de zéatine et de 0,01~0,2 mg/l d'IAA ; et
    (iii) un mélange de 0,5∼10 mg/l de zéatine et de 0,01~0,2 mg/l de NAA.
  11. Procédé selon la revendication 1, dans lequel l'étape (e) suivante est comprise en plus après l'étape (d) : (e) transfert des pousses dans un milieu de formation de racines, puis culture des pousses transférées pour former des racines.
  12. Procédé selon la revendication 1, dans lequel le gène cible est un gène impliqué dans le mécanisme de défense des plantes ou un gène impliqué dans la biosynthèse de métabolites utiles.
  13. Procédé selon la revendication 12, dans lequel le gène impliqué dans le mécanisme de défense des plantes est un gène codant pour une protéine choisie dans le groupe constitué par la protéine d'inactivation des ribosomes (RIP), la carboxyl-méthyltransférase de l'acide jasmonique, la tréhalose synthase, la défensine 1.2 des plantes, la thionine synthase, la glucanase, la chitinase, la phénylalanine ammonia-lyase, la chalcone synthase, la glutathione-S-transférase, l'anthranilate synthase, une protéine de stockage, la calmoduline, la tryptophane synthase, l'inhibiteur II des protéinases, l'acide nitrique synthase, la cystémine, l'acide gras peroxydase, l'oxyde d'allène synthase, le facteur de transcription de l'interaction piment-PMMV 1 (PPI1), le facteur de transcription du domaine WRKY, une protéine sensible aux pathogènes, et une protéine d'enveloppe virale.
  14. Procédé selon la revendication 13, dans lequel le gène codant pour la protéine d'enveloppe virale est choisi dans le groupe constitué par le gène TMV-CP, CMV-CP et PepMoV-CP.
  15. Procédé selon la revendication 12, dans lequel le gène impliqué dans la biosynthèse de métabolites utiles est choisi dans le groupe constitué par les gènes impliqués dans la biosynthèse de la tannine, la sinapine, la saponine, l'allicine, la spinosine, l'acide cinnamique, un flavonoïde, un terpénoïde, la catéchine, la vitamine, la pénicilline, l'indole, l'insuline, la prostaglandine, le taxol, l'alisol, le ricin, un caroténoïde et la capsicine.
  16. Procédé selon la revendication 1, dans lequel le piment est du genre Capsicum.
  17. Procédé selon la revendication 16, dans lequel ledit genre Capsicum est Capsicum annuum L.
  18. Procédé selon la revendication 17, dans lequel ledit Capsicum annuum L. est choisi dans le groupe constitué par le piment chili (Capsicum annuum L. var. acuminatum), le piment doux ou poivron (Capsicum annuum L. var. grossum), le piment conique (Capsicum annuum L. var. conoides), le piment cerise (Capsicum annuum L. var. cerasiforme), le piment à bouquet rouge (Capsicum annuum L. var. fasciculatum) et le pili-pili (Capsicum annuum L. var. longum).
EP04748401.9A 2004-03-12 2004-07-09 Procede pour produire des piments transgeniques par callogenese Expired - Lifetime EP1722624B1 (fr)

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PCT/KR2004/001686 WO2005086993A1 (fr) 2004-03-12 2004-07-09 Procede pour produire des piments transgeniques par callogenese

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CN102286526B (zh) * 2011-08-12 2013-01-16 江苏省农业科学院 一种快速获得辣椒转基因植株的方法
CN110305884B (zh) * 2019-08-05 2022-11-04 云南省烟草农业科学研究院 一种提高烟草叶片茉莉酸含量的基因NtAOS1及其克隆方法与应用
CN116656724A (zh) * 2023-07-06 2023-08-29 上海迈其生物科技有限公司 一种的生菜遗传转化方法
CN116724894B (zh) * 2023-08-09 2023-10-20 山东寿光蔬菜种业集团有限公司 一种辣椒花药培养中减轻外植体褐化的方法
CN117561981A (zh) * 2024-01-17 2024-02-20 浙江大学海南研究院 一种辣椒的愈伤组织再生体系建立方法

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